Project Summary/Abstract The overarching goal of this research program is to advance the current technology for biomedical research and drug development. The focus of this study is to develop Redox-assisted Disulfide Direct Conjugation (RDDC) as a new bioconjugation technology. Cysteine disulfide is a post-translational modification (PTM) that plays important structural and functional roles in proteins. However, current methods for cysteine disulfide conjugation and detection all involve reduction of the disulfides prior to functionalization. Such operation erases the redox information in the protein and eliminates the possibility of differentiating cysteine disulfides from free cysteines. There is no method available for targeting cysteine disulfides directly. Through this project, we will provide a new method for direct functionalization of cysteine disulfides without cross-reacting with free cysteines or compromising the stability of the targeted proteins. It can also be used to detect novel disulfide PTMs. More broadly, this method may address the long-standing selectivity issue in protein functionalization that currently relies on kinetic alkylation or protein engineering despite significant limitations. We will measure the performance of RDDC and demonstrate its utility by homogenous antibody-drug conjugate (ADC) synthesis. This project will also lay the foundation for the future development of chemoproteomic probes for new PTM identification and redox profiling.